The invention relates to non-human mutant mammals deficient in endogenous Sigma receptors, to the cells derived from said mutant animals, and to their application. The invention also relates to a homologous recombination vector with positive-negative selection useful for generating said non-human mutant mammals deficient in endogenous Sigma receptors.
Sigma receptors are binding sites with an affinity for different ligands, some of which are pharmaceuticals of diverse activity. Initially, the interest for Sigma receptors was due to their potential role in the actions of anti-psychotic pharmaceuticals as they showed a high affinity for typical neuroleptic agents such as haloperidol or other compounds causing psychomimetic activity, such as N-allylnormetazocine (SKF-10047). Later on it was discovered that Sigma receptors can be involved in other physiological mechanisms.
Two subclasses of Sigma receptors have been identified, known as type-1 and type-2 Sigma receptors, which can be differentiated by their pharmacological profile, function and molecular size. Both types show high to moderate affinity for typical neuroleptic agents, particularly haloperidol. However, type-1 Sigma receptors show a high affinity for (+)-pentazocine, (+)-SKF10047 and other (+)-benzo-morfanes, while type-2 Sigma receptors show a low affinity for these compounds. Type-1 Sigma receptors have a typical molecular mass of 25 kDa, while the molecular mass of type-2 Sigma receptors is 18-21 kDa (in both cases determined by photoaffinity marking).
Type 1 Sigma Receptor
The type-1 Sigma receptor, hereinafter referred to as Sigma-1 receptor, is a non-opiaceous type receptor expressed in numerous adult mammal tissues (central nervous system, ovary, testicle, placenta, adrenal gland, spleen, liver, kidney, gastrointestinal tract, etc.) as well as in embryo development from its earliest stages, and is apparently involved in a large number of physiological functions. Its high affinity for various pharmaceuticals has been described, such as for SKF-10047, (+)-pentazocine, haloperidol and rimcazole, among others, known ligands with analgesic, anxiolytic, antidepressive, antiamnesic, antipsychotic and neuroprotective activity, so that the study of the Sigma-1 receptor is of great interest in pharmacology in view of its possible physiological role in processes related to analgesia, anxiety, addiction, amnesia, depression, schizophrenia, stress, neuroprotection and psychosis. See
Kaiser C., Pontecorvo M. J. & Mewshaw R. E. (1991) Sigma receptor ligands: function and activity. Neurotransmissions 7 (1): 1-5 (hereinafter Kaiser); Walker J. M., Bowen W. D., Walker F. O., Matsumoto R. R., De Costa B. & Rice K. C. (1990) Sigma receptors: biology and function. Pharmacological Reviews 42 (4): 355-402 (hereinafter Walker); and Bowen W. D. (2000) Sigma receptors: recent advances and new clinical potentials. Pharmaceutica Acta Helvetiae 74: 211-218 (hereinafter Bowen).
However, the actual role carried out by the Sigma-1 receptor is still unknown and enigmatic. In fact, its specific endogenous ligand is not known, although it is believed that it interacts with endogenous human steroids such as progesterone and testosterone.
The cDNA sequence encoding the Sigma-1 receptor in guinea pigs, the cDNA and Sigma-1 receptor gene in humans, the cDNA sequence and Sigma-1 receptor gene in mice and the cDNA encoding the Sigma-1 receptor in rats have all been described.
The first work to obtain a gene sequence encoding a Sigma-1 receptor is due to Hanner et al. (see Hanner M., Moebius F. F., Flandorfer A., Knaus H. G., Striessing J., Kempner E. & Glossmann H. (1996) Purification, molecular cloning, and expression of the mammalian Sigma-1 binding site. Proceedings of the National Academy of Sciences USA 93: 8072-8077, hereinafter Hanner).
In this work, using the specific binding characteristics of this receptor to certain radioactively marked compounds, such as (+)[3H]Pentazocine, a protein first and then a cDNA clone encoding it were obtained. The clone was isolated from a cDNA genomic bank prepared from Guinea pig (C. porcellus) liver mRNA (cDNA with 1857 base pairs, GenBank database, access code Z66537).
Next, based on this cDNA sequence, several work groups proceeded to successively clone by sequence homology the human cDNA (see Kekuda R., Prasad P. D., Fei Y.-J., Leibach F. H. & Ganapathy V. (1996) Cloning and functional expression of the human type 1 Sigma receptor (hSigmaR1). Biochemical and Biophysical Research Communications 229: 553-558, hereinafter Kekuda) (cDNA with 1653 base pairs, GenBank database, access code: HSU75283), the mouse cDNA and gene (see Seth P., Leibach F. H. & Ganapathy V. (1997) Cloning and structural analysis of the cDNA and the gene encoding the murine type I sigma receptor. Biochemical and Biophysical Research Communications 241: 535-540, hereinafter Seth, 1997) (cDNA with 1567 base pairs, GenBank database, access code: AF030198; gene with 6973 base pairs, GenBank database, access code: AF030199), the rat cDNA (see Seth P., Fei Y.-J., Li H.-W., Huang W., Leibach F.-H. & Ganapathy V. (1998) Cloning and functional characterization of a receptor from rat brain. Journal of Neurochemistry 70: 922-931, hereinafter Seth, 1998) (cDNA with 1582 base pairs, GenBank database, access code: AF004218) and the human gene (see Prasad P. D., Hui W. L., Fei Y.-J., Ganapathy M. E., Fujita T., Plumley L. H., Yang-Feng T.-L., Leibach F.-H. & Ganapathy V. (1998) Exon-intron structure, analysis of promoter region, and chromosomal localization of the human Type I receptor gene. Journal of Neurochemistry 70: 443-451, hereinafter Prasad) (gene described in three DNA fragments respectively containing the promoter (3589 base pairs, GenBank database, access code: AF001975), the exon 1, 2 and 3 (757 base pairs, GenBank database, access code: AF001976) and the exon 4 (1630 base pairs, GenBank database, access code: AF001977)).
An analysis of the homology among these sequences, both on the basis of nucleotides and amino acids, has made manifest the high degree of homology among them. Knowledge of these sequences can be used to develop animal models intended to study the physiology and pathology associated to alterations in Sigma receptors and the effect of pharmaceuticals potentially useful for treating or preventing pathologies associated to alterations in said receptors or in which said receptors are involved.
Type-2 Sigma Receptor
The type-2 Sigma receptor, hereinafter referred to as the Sigma-2 receptor, is a receptor expressed in numerous adult mammal tissues (nervous system, immune system, endocrine system, liver, kidney, etc.). Sigma-2 receptors can be components in a new apoptosis route that may play an important role in regulating cell proliferation or in cell development. This route seems to consist of Sigma-2 receptors joined to intracellular membranes, located in organelles storing calcium, such as the endoplasmic reticulum and mitochondria, which also have the ability to release calcium from these organelles. The calcium signals can be used in the signalling route for normal cells and/or in induction of apoptosis.
Agonists of Sigma-2 receptors induce changes in cell morphology, apoptosis in several types of cell lines and regulate the expression of p-glycoprotein mRNA, so that they are potentially useful as antineoplasic agents for treatment of cancer. In fact, Sigma-2 receptor agonists have been observed to induce apoptosis in mammary tumour cell lines resistant to common antineoplasic agents that damage the DNA.
In addition, agonists of Sigma-2 receptors enhance the cytotoxic effects of these antineoplasic agents at concentrations in which the agonist is not cytotoxic. Thus, agonists of Sigma-2 receptors can be used as antineoplasic agents at doses inducing apoptosis or at sub-toxic doses in combination with other antineoplasic agents to revert the resistance to the drug, thereby allowing using lower doses of the antineoplasic agent and considerably reducing its adverse effects. Agonists of Sigma-2 receptors can also be used in image diagnosis of cancer as agents for non-invasive visualisation of tumours and their metastasis with imaging diagnostic techniques, such as SPECT or PET. Therefore, Sigma-2 receptors constitute targets of great interest for cancer-fighting tools.
Antagonists of Sigma-2 receptors can prevent the irreversible motor side effects caused by typical neuroleptic agents. In fact, it has been found that antagonists of Sigma-2 receptors can be useful as agents for improving the weakening effects of delayed dyskinesia appearing in patients due to chronic treatment of psychosis with typical antipsychotic drugs, such as haloperidol. Sigma-2 receptors also seem to play a role in certain degenerative disorders in which blocking these receptors could be useful. For more information on Sigma-2 receptors, see Bowen.
Sigma-2 receptors seem, to be involved in numerous physiological functions; however, as with Sigma-1 receptors the actual role played by Sigma-2 receptors is not entirely known, so that the need exists to increase their understanding. An alternative for contributing to the understanding of the physiological functions in which said Sigma-2 receptors are involved consists of developing animal models intended for studying the physiology and pathology in which the receptors may be involved and the effect of drugs potentially useful in the treatment and prevention of the pathologies in which said receptors are involved.
“Knockout” Technique
Genetic manipulation of mammals allows obtaining transgenic animals expressing a specific gene, or alternatively having a gene inactivated by a specific mutation (“knockout” or mutant animals). In both cases their potential use for designing experimental models in laboratory animals allowing analysing the function of a given gene in vivo is obvious.
The “knockout” technique for generating mutant animals is well established and is the object of many publications. By way of example, the following U.S. patents may be cited: U.S. Pat. Nos. 5,464,764, 5,487,992, 5,627,059, 5,631,153, 6,194,633, 6,207,876, 6,239,326, 6,245,963, 6,245,965 and 6,252,132, among others.
Lack of expression of a gene can confer a new phenotype to a mutant animal. Depending on the gene that is not expressed by the animal, it can become more or less susceptible to a given pathologic alteration. These mutant animals are valuable models for an in vivo study of the role of the gene, as well as in the study of compounds that could be potentially useful in treatment or prevention of pathologies related to the lack or expression or ineffective expression of the product of this gene.
Although Sigma receptors have been described to show an affinity for compounds with diverse pharmacological activity, the actual role played by these receptors is currently unknown, so that it is necessary to generate animal models to study in vivo the role played by endogenous Sigma receptors. A mutant animal deficient in endogenous Sigma receptors would contribute to defining the role played in vivo by these receptors and would allow designing and evaluating compounds potentially interesting for treating processes related to the null or ineffective expression of these Sigma receptors. The invention provides a solution to this current need.